Phytase-producing bacteria

ABSTRACT

This invention relates to the isolation of thermophilic phytase-producing microorganisms, method for producing phytase using such microorganisms, phytase obtained therefrom, and usage of the phytase to hydrolyze phytic acid or phytate. In particular, this invention relates to phytase-producing microorganisms, which belong to Streptomyces sp., Pseudonocardia sp. or Microbispora sp., and which produce phytase available for recovery in an efficient and practical manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of parent application Ser.No. 09/038,705, filed Mar. 9, 1998, the disclosure of which parentapplication Ser. No. 09/038,705 is incorporated herein by reference.Also, this application through parent application Ser. No. 09/038,705claims benefit under 35 U.S.C. §119(e) to earlier filed provisionalapplication Ser. No. 60/057,780, filed Sep. 8, 1997.

FIELD OF INVENTION

This invention relates to the isolation of thermophilicphytase-producing microorganisms, method for producing phytase usingsuch microorganisms, phytase obtained therefrom, and usage of thephytase to hydrolyze phytic acid or phytate. In particular, thisinvention relates to phytase-producing microorganisms, which belong toStreptomyces sp., Pseudonocardia sp. or Microbispora sp., and whichproduce phytase available for recovery in an efficient and practicalmanner.

BACKGROUND OF THE INVNETION

Phytic acid (myoinositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) isknown as one of the major component of plant-derived food. It is theprimarily source of inositol and the main storage form of phosphorus inplant seeds that are used as animal feed ingredients (oilseed meals,cereal grains, and legumes). Approximately 75% of the total phosphorusin cereals, legumes and seeds exist as phytic acid phosphorus. When oneor more of the acidic protons of the phophate groups in phytic acid arereplaced by a counterion, the compound is usually referred to as aphytate salt. The name phytin is used for the calcium-magnesium salt ofphytate derived from plant seeds (a discontinued product of Ciba-Geigy).

Phytic acid plays an important role in the dormacy and germinationstages of plant seeds. It was believed that phosphorus was liberated ongermination and incorporated into ATP. Recent studies have estblishedthe role of inositol phosphate intermediates in the transport ofmaterials into cells and their role in transport as secondary messengersand in signal transduction in plant and animal cells is a very activearea of research.

There are many applications of phytic acid, including industrial use asa corrosion inhibitor on metals, a rust remover and an additive tolubricating greases, use as a food additive, and medical applications,including use in the prevention of dental caries, use as an imagingagent for organ scintography and an X-ray enhancement contrasting agent,use as a hypacholestromic agent, use to reduce gastric secretion fortreatment of gastritis, gastroduodenitis, gastric duodenal ulcers anddiarrhea, use as an antidote for toxic metal absorption, therapeuticuses in the prevention and dilution of calcium deposits associated withvarious diseases and for reducing calcium concentration in urine (thuschecking the formation of renal calculi), use as a preventive agentagainst severe poisoning with pressurized oxygen and preventing thirstduring exercise, use as a taste-improving agent in orally administeredantibiotics, and use in the treatment of multiple sclerosis (see U.S.Pat. No. 5,217,959 issued to Robert Sabin). For further discussions ofindustrial applications of phytic acid, see Graf, JAOCS 60, 1861-1867,1983.

Phytic acid may be prepared in pure form from various plant sources,such as wheat, corn, soybeans, sesame seeds, peanuts, lima beans,barley, oats, wild rice and sunflower seeds, and it can be extractedwith dilute hydrochloric acid at room temperature, precipitated withvarious reagents including ferric chloride, bicarbonates, potassiumhydroxide, sodium hydroxide, ammonium hydroxide, calcium hydroxide,magnesium hydroxide or alcohol. It is then further purified byconventional chemical techniques. Hydrolyis of phytic acid and phytatesmay be carried out by partial acid or basic hydrolysis or by hydrolysisusing phytase, and the resultant products include phosphate, inositoland various inositol phosphate intermediates.

Phytic acid phosphorus has anti-nutritive properties as it isessentially poorly metabolized by monogastric animals, such as pourltryand swine, because these animals produce little or no phytase in theirdigestive tracts. In addition, phytic acid forms complexes with proteinsand divalent cations, such as calcium, iron, zinc, magnesium, manganese,copper and molybdenum. Phytic acid also binds to starch and influencesthe digestibility and solubility of starch. Phytic acid excreted in themanure of feed animals is enzymatically hydrolyzed by soil and watermicroorganisms. The released phosphorus is transported into rivers andlakes and if introduced in high qunatities causes eutrophication. Theanti-nutritive properties and its valus as a possible pohosphorussource, have stimulated researchers to develop a method to remove phyticacid in a manner that is economically competitive wuth mineralsupplementation.

Hydrolyzing phytic acid is thought to be a useful way of increasing thenutritional value of many plant foodstuffs. The enzymes that catalyzethe conversion of phytic acid to inositol and inorganic phosphate areknown as phytases. Phytase is found to be distributed in the seeds andpollens of plants, and also some microorganisms. The mass production ofphytase from plant origin is not economic since preliminary treatment isnecessary and the production procedure becomes time-comsuming,troublesome and expensive. Therefore, the production of phytase frommicrobial origin is of greater potential in development. The feeding ofmicrobial phytase to monogastric animals alter the phytic acid complexesand increase the bioavailability of phosphorus, calcium and probablyproteins to monogastric animals.

The research of phytase spans more than 87 years from its discovery bySuzuki et al. (Tokyo Imp. Univ. Coll. Agr. Bull., 7: 503-512, 1907)until its commercialization in Europe in 1993-1994 by Gist-brocades. Theinternational Union of Biochemistry (1979) lists two phytase: a3-phytase, EC 3.1.3.8, which hydrolyzes the ester bond at the 3-positionof myoinositolhexakus phosphate to D-myoinositol1,2,4,5,6-pentakisphosphate + orthophosphate, and a 6-phytase, EC3.1.3.26, which first phdrolyzes the 6-position of myoinositolhexakusphosphate to D-myoinositol 1,2,3,4,5-pentakisphosphate + orthophosphate.Subsequent ester bonds in the substrate are hydrolyzed at differentrates. The 6-phytase dephosphorylates phytic acid completely, whereasthe aforesaid 3-phytase does not hydrolyze the phosphomono ester.

Currently, phytase-producing microorganisms include bacteria, such asBacillus subtilis and Escherichia coli; yeasts, such as Saccharomycescerevisias and Schwannoiomyces castellii; and fungi, such as Aspergillusniger, A. oryzae, A. ficuum, Penicillium simplicissimum.

Yeasts produce phytase intracellularly and, hence, it is difficult andless efficient to recover phytase with a high yield.

Of all the microorganisms surveyed, Aspergillus niger (syn. A. ficuum)NRRL 3135 produces phytase extracelllularly, and the phytase producedthereby is known to be most active and has been commercialized. Themicroorganism is subjected to solid state fermentation (SSF) togetherwith cereal grains, legume beans or foodstuffs so as to substantiallyremove or reduce phytic acid therefrom.

A thorough review of the research and development of phytase is providedby Rudy J. Wodzinski et al., in "Phytase," Advances in AppliedMicrobiology, Vol. 42, p. 263-303, 1996, and other relevant docuementaryreferences are cited in the Reference List accompanying theSpecification.

However, since during fermentation, the temperature within the fermenternormally will rise to 55° C. or higher, the efficiacy of removing phyticacid by microbial fermentation using non-thermophilic microorganisms issignificantly decreased. In addition, fungal cells grow slowly andhence, it takes longer time (about 10 days) for the fungal cells toproduce phytase having high enzymatic activity.

There thus still exists a need to produce phytase by a phytase-producingmicroorganism which is thermophilic and which maintains the ability toremove phytic acid during solid state fermentation. The industrialapplication of such a phytase-producing microorganism is promising asthe phytase produced thereby is thermostable and fermentation using thesame is efficient and energy saving.

SUMMARY OF THE INVENTION

An object of this invention is to isolate a thermophilic microorganismwhich produced phytase extracellularlly and which can be used inlarge-scale solid state fermentation for the industrial production ofphytase.

Another object of this invention is to industrially produce phytase byusing said thermophilic microorganism in large-scale solid statefermentation.

Heretofore, it has not been reported that actinomycetes produce phytase.

The present invention screened and obtained 14 microbial isolates whichare thermophilic and produce phytase extracellularlly. These isolatesinclude Streptomyces thermoviolaceus subsp. thermoviolaceus CCRC 12493,Streptomyces thermodiastaticus CCRC 12492 and Streptomycesthermoviolaceus subsp. thermoviolaceus CCRC 12639, the three isolatesbeing microorganisms listed in the Catalogue of Bacteria, Bacteriophages& Recombinant DNA Materials published by the Food Industry Research andDevelopment Institute (FIRDI), Hsin-Chu, Taiwan, R.O.C., isolate T16-4(Streptomyces thermonitrificans), isolate T17-1 (Streptomycesthermonitrificans), isolate T17-2 (Streptomyces thermovulgaris), T17-4(Pseudonocardia thermophila), isolate T17-6 (Pseudonocardiathermophila), isolate T19-3 (Streptomyces sp.), isolate T24-3(Streptomyces diastaticus), isolate T26-6 (Streptomyces sp.), isolateT28-1 (Streptomyces sp.), isolate T29-1 (Microbispora sp.), and isolateT45-l (Streptomyces aurantiogriseus), the eleven isolates having beendeposited in the China Center for Type Culture Collection (CCTCC), WuhanUniversity, Luo Jia Shan, Wuhan, the People's Republic of China underthe Budapest Treaty for the purposes of patent applications on Sep. 29,1997 with the accession numbers of CCTCC M97007, CCTCC M97008, CCTCCM97009, CCTCC M970010, CCTCC M970011, CCTCC M970012, CCTCC M970013,CCTCC M970014, CCTCC M970015, CCTCC M970016 and CCTCC M970017,respectively. Isolates T16-4, T24-3 and T45-l were also deposited in theFIRDI for the purpose of patent application on Mar. 6, 1997 with theaccession numbers of CCRC 910076, CCRC 910075 and CCRC 910074,respectively.

Features and advantages of the present invention will become apparent inthe following detailed description of the examples, with reference tothe accompanying drawings, of which:

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a graph showing the influence of pH to the enzyme activity ofphytase produced by isolate Tl7-2; and

FIG. 2 is a graph showing the influence of temperature to the enzymeactivity of phytase produced by isolate T17-2.

EXAMPLES

A. Methods and Materials:

Medium: The formulation of the phytase screen medium (PSM) is shownbelow:

    ______________________________________                                               1.5%        glucose                                                           0.5%        NH.sub.4 NO.sub.3                                                 0.05%       KCl                                                               0.05%       MgSO.sub.4 · 7H.sub.2 O                                  0.001%      FeSO.sub.4 · 7H.sub.2 O                                  0.001%      MnSO.sub.4 · 7H.sub.2 O                                  0.05%       Ca-phytate                                                        2.0%        agar                                                              adjust pH to 5.5                                                       ______________________________________                                    

I. Cultivation of phytase-producing microorganisms:

The microorganism to be screened is cultivated in TSB medium (15 g/Ltryptone, 5.0g/L soybtone, 5.0 g/L NaCl, 1.0 L distilled water) at 45°C. overnight. 0.1 ml of the cultivated mcroorganism is collected andinoculated into 50 ml PSM broth, allowing the microorganism to grow at45° C. for 4 days with agitation at a rate of 125 rpm.

II. Screening of phytase-producing microorganisms:

Isolation of the phytase-producing microorganism is carried out by:sampling soil from the south part of Taiwan, subjecting the soil samplesto HV medium (1 g/L humic acid, 0.5 g/L Na₂ HPO₄, 1.7 g/L KCl, 0.05 g/LMgSO₄.7H₂ O, 0.02 g/L CaCO₃, B-vitamin, agar 18 g/L), and cultivatingthe samples at 50° C. to screen isolates which produce phytaseextracellularly. Streptomytes deposited in the FIRDI, Taiwan, R.O.C. arealso screened concomitantly in a same manner to collect those strainswhich are phytase-producing.

III. Detection of the enzymatic activity of phytase:

The phytase activity is detected by the following method:

1. Inoculate the isolate into a 50 ml PSM broth (substituting 0.05%Ca-phytate with 0.01% Na-phytate), cultivate the isolate at 45° C. for 4days, and centrifugate the isolate at 3000 rpm for 5 minutes, thesupernatant being collected as an enzyme solution;

2. Preparation of enzyme substrate: dissolve 1.64 g of Na-phytate in0.25 M sodium acetate buffer (pH 5.5).

dissolve 1.64 g of Na-phytate in 0.25M glycine-HCl buffer (pH 2.5).

3. 1 ml enzyme solution is added with 1 ml enzyme substrate, allows themixture to react at 55° C. for 60 minutes, and 10% trichloroacetic acid(TCA) is added into the mixture to stop reaction. Adds 2 ml colordeveloping agent (3.66 g FeSo₄.7H₂ 0, 0.5 g ammonium heptamolybdatetetrahydrate, 1.6 ml conc. H₂ SO₄, adding H₂ O to 50 ml) into themixture and detect the absorbance at 750 nm. The calibration curve ismade with use of NaH₂ PO₄. A unit of phytase activity is defined as 1μmol phosphate produced by 1 ml enzyme solution per minute.

B. Results:

The soil samples are cultured in PSM at 50° C. for 7 days, and microbialcolonies which are surrounded with a clear zone on the culture platesare isolated and screened. 150 microbial isolates which are derived fromthe soil samples of the south part of Taiwan and 30 actinomycete strainsdeposited in the FIRDI are cutivated in PSM to screen microorganismswhich are thermophilic and produce phytase extracellularly. Thesemicroorganisms are inoculated into 50 ml PSM broth and grow at 45° C.for 4 days with agitation at at rate of 125 rpm. The microbial culturesare centrifugated at 3000 rpm and the supernatants are collected. Thephytase activity is respectively detected under pH 5.5 at a temperatureof 550C and under pH 2.5 at a temperature of 55° C.

The enzyme activity of phytase produced by the obtained 14 isolates andthe sources of these isolates are shown in Table 1.

The detected enzyme activity of phytase produced by isloate T45-1 is8.9×10⁻⁴ U/ml under pH 5.5 at a temperature of 55° C. and 2.5×10⁻⁴ U/mlunder pH 2.5 at a temperature of 55° C., respectively.

The detected enzyme activity of phytase produced by isloate T24-3 is5×10⁻⁴ U/ml under pH 5.5 at a temperature of 55° C. and 1×10⁻⁵ U/mlunder pH 2.5 at a temperature of 55° C., respectively.

The detected enzyme activity of phytase produced by isloate T16-4 is3.8×10⁻⁴ U/ml under pH 5.5 at a temperature of 55° C. and 1.1×10⁻⁵ U/mlunder pH 2.5 at a temperature of 55° C., respectively.

The detected enzyme activity of phytase produced by Streptomycesthermoviolaceus subsp. thermoviolaceus CCRC 12493, is 1.8×10⁻³ U/mlunder pH 5.5 at a temperature of 55° C. and 2.5×⁻⁴ U/ml under pH 2.5 ata temperature of 55° C., respectively.

The detected enzyme activity of phytase produced by Streptomycesthermodiastaticus CCRC 12492 is 8.2×10⁻⁴ U/ml under pH 5.5 at atemperature of 55° C. and 3.1×10⁻⁴ U/ml under pH 2.5 at a temperature of55° C., respectively.

The detected enzyme activity of phytase produced by Streptomycesthermoviolaceus subsp. thermoviolaceus CCRC 12639 is 1.1×10⁻⁴ U/ml underpH 5.5 at a temperature of 55° C. and 9.3×³¹ 5 U/ml under pH 2.5 at atemperature of 55° C., respectively.

Further ioslates are obtained from the same screening procedure and theyare isolates T17-1, T17-2, T17-4, T17-6, T19-3, T26-6, T28-1 and T29-3.

The detected enzyme activity of phytase produced by isloate T17-2 is2.2×10⁻³ U/ml under pH 5.5 at a temperature of 55° C. and 2.8×10⁻⁴ U/mlunder pH 2.5 at a temperature of 55° C. It is T17-2 is 5.7 U/ml under pH5.5 at a temperature of 55° C. It is found that when the pH andtemperature for the enzyme activity analysis are varied, the producedphytase is most active at pH 5.5 (FIG. 1) and the optimal temperaturefor the enzyme is in the range from 55° C. to 60° C., with 90% activitybeing maintained at 65° C. (FIG. 2). In addition, when isolate T17-2 iscultivated in media supplemented with different carbon and nitrogensources, the produced phytase is most active when the isolate iscultivated in a medium containing 1.5% glucose and 0.5% NH₄ NO₃ (Table2).

The obtained microbial isolate T45-1 has the following taxonomicfeatures:

1. gram-positive,

2. aerial mycelium,

3. the spore surface is smooth and the spore chain is in spiral form,

4. the cellular hydrolysates include L,L-diaminopimelic acid and nospecific sugar is present on the cell wall of the microorganism,

5. colonies with wrinkled surface are present on the HV plate.

Based on the above features, isolate T45-1 is determined and classifiedas a Streptomyces auranthigriseus according to the Bergey's Manual ofSystematic Bacteriology.

The obtained microbial isolate T24-3 has the following taxonomicfeatures:

1. gram-positive,

2. aerial mycelium,

3. the spore surface is smooth and the spore chain is in spiral

4. the cellular hydrolysates include L,L-diaminopimelic acid and nospecific sugar is present on the cell wall of the microorganism,

5. growth temperature: 30° C. to 49° C.

6. colonies carrying grey-black spores on the surface are present on theHV plate.

Based on the above features, isolate T24-3 is determined and classifiedas a Streptomyces diastaticus according to the Bergey's Manual ofSystematic Bacteriology.

The obtained microbial isolate T16-4 has the following taxonomicfeatures:

1. gram-positive,

2. aerial mycelium,

3. the spore surface is smooth and the spore chain is in spiral form,

4. the cellular hydrolysates include L,L-diaminopimelic acid and nospecific sugar is present on the cell wall of the microorganism,

5. growth temperature: 30° C. to 49° C.

6. colonies carrying white spores are present on the HV plate.

Based on the above features, isolate T16-4 is determined and classifiedas a Streptomyces thermmonitrificans according to the Bergey's Manual ofSystematic Bacteriology.

The obtained microbial isolate T17-1 has the following taxonomicfeatures:

1. gram-positive,

2. aerial mycelium,

3. the spore surface is smooth and the spore chain is in spiral form,

4. the cellular hydrolysates include L,L-diaminopimelic acid and nospecific sugar is present on the cell wall of the microorganism,

5. growth temperature: 30° C. to 49° C.

Based on the above features, isolate T17-1 is determined and classifiedas a Streptomyces thermmonitrificans according to the Bergey's Manual ofSystematic Bacteriology.

The physiological and biochemical characters of the eleven isolatesobtained in this invention are analysed and listed in Table 3, and acomparison of the same with reference strains, which are known standardmicroorganism strains deposited in the FIRDI and the scientific names ofwhich are listed in Table 4, is made and listed in Table 5. From thecomparison with the reference strains, the taxons of these isolates aredetermined and listed in Table 6.

With respecpect to isolates T17-4 and T17-6, since the two isolates areanalogous to Pseudonocardia theremophila in terms of morphology andfatty acid contents (see Tables 7 and 8), they are subjected to G+Ccontent analysis and DNA hybridization test. The two isolates are foundto respectively have a G+C percentage of 74% and 75.2%, and their DNAhybridization results show a homology greater than 98%. Therefore,isolates T17-4 and T17-6 are classified as Pseudonocardia theremophila.

From the above teachings, it is apparent that various modifications andvariations can be made without departing from the psirit and scope ofthe present invention. It is therefore to be understood that thisinvention may be practiced otherwise than as specifically described.

                  TABLE 1                                                         ______________________________________                                        The phytase activities and isolation sources                                  of the obtained phytase-producing isolates.                                   ISOLATE NO.                                                                            PHYTASE ACTIVITY (U/ml) AT 55° C.                                                            SOURCE                                         ______________________________________                                        T17-2    2.2 × 10.sup.-3 goat manure                                    T19-3    2.1 × 10.sup.-3 soil                                           T29-1    1.9 × 10.sup.-3 soil                                           CCRC12639                                                                              1.1 × 10.sup.-4 FIRDI                                          CCRC12493                                                                              1.8 × 10.sup.-3 FIRDI                                          T28-1    9.1 × 10.sup.-4 soil                                           T45-1    8.9 × 10.sup.-4 soil                                           T17-1    8.8 × 10.sup.-4 goat manure                                    T26-6    8.5 × 10.sup.-4 soil                                           CCRC12492                                                                              8.2 × 10.sup.-4 FIRDI                                          T24-3      5 × 10.sup.-4 soil                                           T16-4    3.8 × 10.sup.-4 soil                                           T17-6    3.1 × 10.sup.-4 goat manure                                    T17-4    3.6 × 10.sup.-4 goat manure                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        The phytase activities of the T17-2 incubated                                 at different carbon and nitrogen sources.                                     C source  Activity (10.sup.-3                                                                       N source   Activity (10.sup.-3                          (1.5%)    U/ml at 55° C.)                                                                    (0.5%)     U/ml at 55° C.)                       ______________________________________                                        glucose   5.6         NH.sub.4 NO.sub.3                                                                        5.6                                          sucrose   3.5         yeast extract                                                                            1.3                                          xylose    1.2         polypeptone                                                                              1.9                                          arabinose 1.2         (NH.sub.4).sub.2 SO.sub.4                                                                0.8                                          lactose   0.5         KNO.sub.3  2.5                                          maltose   1.5         urea       0.9                                          corn starch                                                                             0.8         glycine    0.5                                          soluble starch                                                                          2.8                                                                 mannitol  0.5                                                                 ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    The physiological and biochemical character analyses of the obtained          phytase-producing isolates.                                                          Isolate                                                                Analysis items                                                                       T16-4                                                                             T17-1                                                                             T17-2                                                                             T17-4                                                                             T17-6                                                                             T19-3                                                                             T24-3                                                                             T26-6                                                                             T28-1                                                                             T29-1                                                                             T45-1                          __________________________________________________________________________    Utility of                                                                    Arabinose                                                                            -   -   +   Nd. Nd. -   -   +/- -   +   +                              Cellulose                                                                            -   -   -   +   +   -   -   -   -   -   -                              Fructose                                                                             -   +   -   -   -   -   -   -   -   -   +                              Inositol                                                                             +   +   +   +   +   +/- +/- +/- -   -   +                              Lactose                                                                              +   +   -   +   +   -   -   -   +   +   +                              Mannitol                                                                             -   +   -   -   -   +   +   +   +   +   +                              Raffinose                                                                            -   -   -   +   +   -   -   +   +   -   +                              Rhamnose                                                                             -   -   -   +   +   -   -   -   +   +   +                              Salicin                                                                              -   +   -   -   +   -   -   -   -   -   +                              Sucrose                                                                              -   -   -   -   -   -   -   -   -   +   +                              Xylose -   +   +   -   -   +   +   +   +   +   +                              Degradation of                                                                Adenine                                                                              -   -   -   -   -   -   -   +   +   -   +                              Caseine                                                                              +   +   +   Nd. Nd. +   +   +   +   +   +                              Esculin                                                                              -   +   -   -   -   -   -   +   +   -   +                              Hypoxanthine                                                                         +   -   -   -   -   -   +   +   +   -   +                              L-tyrosine                                                                           +   +   +   +   +   +   +   +(dark.sup.a)                                                                     +(dark.sup.a)                                                                     -   +                              Xanthine                                                                             +   -   -   -   -   -   -   -   -   -   +                              Lysozyme                                                                             -   -   -   -   -   -   -   -   -   -   -                              Urease -   -   -   +   +   -   -   -   -   -   -                              __________________________________________________________________________     Nd: not determine                                                             .sup.a : producing melanin in broth                                      

                  TABLE 4                                                         ______________________________________                                        Scientific name list of the Standard strains                                  deposited in the FIRDI.                                                       Standard strain no.                                                                          Scientific name                                                ______________________________________                                        CCRC10483 = ATCC23916                                                                        S. griseofuscus                                                CCRC11465 = ATCC19743                                                                        S. collinus                                                    CCRC12034 = ATCC29251                                                                        S. longwoodensis                                               CCRC12068 = ATCC2S453                                                                        S. flocculus                                                   CCRC12169 = ATCC11874                                                                        S. cinnamoneus                                                 CCRC12492 = NRRL B-5316                                                                      S. thermodiastaticus                                           CCRC12493 = NRRL 12374                                                                       S. thermoviolaceus                                             CCRC12636 = ATCC27472                                                                        S. thermodiastaticus                                           CCRC12638 = ATCC19284                                                                        S. thermovulgaris                                              CCRC12639 = ATCC19283                                                                        S. thermoviolaceus subsp. thermoviolaceus                      CCRC13311 = ATCC15723                                                                        S. poonensis                                                   ______________________________________                                    

    __________________________________________________________________________    The results of biophyical and biochemical character of type strains of        streptomyces.                                                                 __________________________________________________________________________           Type strain                                                            Analysis items                                                                       CCRC10483                                                                           CCRC12034                                                                           CCRC12068                                                                           CRRC11465                                                                           CCRC12169                                                                           CCRC12492                                __________________________________________________________________________    Utility of                                                                    Arabinose                                                                            +     -     +     +     -     -                                        Cellulose                                                                            -     -     -     -     -     -                                        Fructose                                                                             +     -     +     -     -     -                                        Inositol                                                                             -     +     +     +     -     -                                        Lactose                                                                              -     +     +     -     +     +                                        Mannitol                                                                             -     -     +     +     -     +                                        Raffinose                                                                            -     -     -     +     -     -                                        Rhamnose                                                                             -     -     +     -     -     -                                        Silicin                                                                              -     -     +     -     -     -                                        Sucrose                                                                              -     -     -     +     -     -                                        Xylose +     +     -     -     -     +                                        Degradation of                                                                Adenine                                                                              +     +     +     +     -     +                                        Hypoxanthine                                                                         +     +     +     +     +     -                                        L-tyrosine                                                                           +     +     +     +     +     +(dark.sup.a)                            Xanthine                                                                             +     -     +     +     -     -                                        Caseine                                                                              +     +     +     +     -     +                                        Esculin                                                                              +     +     -     -     -     +                                        Lysozyme                                                                             -     -     -     -     +     -                                        Urease -     -     +     -     -     -                                        __________________________________________________________________________                 Type strain                                                      Analysis items                                                                             CCRC12493                                                                           CCRC12636                                                                           CCRC12638                                                                           CCRC12639                                                                           CCRC13311                                __________________________________________________________________________    Utility of                                                                    Arabinose    -     -     +     -     +                                        Cellulose    -     -     -     -     +                                        Fructose     -     -     +     -     -                                        Inositol     -     -     +     +     +                                        Lactose      -     +     -     -     +                                        Mannitol     +     +     +     -     +                                        Raffinose    -     +     -     -     -                                        Rhamnose     -     -     -     -     -                                        Silicin      -     -     -     -     +                                        Sucrose      -     -     -     -     -                                        Xylose       +     +     +     -     +                                        Degradation of                                                                Adenine      +     +     +     +     +                                        Hypoxanthine +     -     -     -     +                                        L-tyrosine   +(dark.sup.a)                                                                       +(dark.sup.a)                                                                       +(dark.sup.a)                                                                       +(dark.sup.a)                                                                       +                                        Xanthine     -     -     -     -     +                                        Caseine      +     +     +     +     -                                        Esculin      +     +     +     +     -                                        Lysozyme     -     -     -     -     -                                        Urease       -     -     -     -     +                                        __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        The identified names of the obtained phytase-producing isolates               Isolate no.    Identified results                                             ______________________________________                                        T16-4          Streptomyces thermonitrificans                                 T17-1          Streptomyces thermonitrificans                                 T17-2          Streptomyces thermovulgaris                                    T17-4          Pseudonocardia thermophila                                     T17-6          Pseudonocardia thermophila                                     T19-3          Streptomyces sp.                                               T24-3          Streptomyces diastaticus                                       T26-6          Streptomyces sp.                                               T28-1          Streptomyces sp.                                               T29-1          Microbispora sp.                                               T45-1          Streptomyces aurantiogriseus                                   ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        The fatty acid content of isolate T17-6.                                      Strain: T17-6                                                                 fatty acid species                                                                            content (%)                                                   ______________________________________                                        15:0 ISO        2.54                                                          16:0 ISO        28.6                                                          16:0            3.8                                                           ISO 17:1 w9c    3.18                                                          17:0 ISO        24.27                                                         17:0 ANTEISO    17.40                                                         17:0            2.76                                                          17:1 w6c        1.71                                                          17:0            2.76                                                          16:1 2OH        3.77                                                          17:0 10 methyl  0.89                                                          18:0 ISO        6.09                                                          18:0            3.45                                                          19:0 ANTEISO    0.63                                                          ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        The fatty acid content of isolate T17-4.                                      Strain: 17-4                                                                  fatty acid species                                                                            content (%)                                                   ______________________________________                                        14:0 ISO        0.4                                                           15:0 ISO        3.76                                                          15:0 ANTEISO    0.34                                                          15:0            0.41                                                          ISO 16:1 ISO H  0.33                                                          16:0 ISO        36.01                                                         16:0            2.71                                                          ISO 17:1 w9c    5.55                                                          17:0 ISO        20.13                                                         17:0 ANTEISO    14.91                                                         17:1 w8c        0.55                                                          17: w6c         3.00                                                          17:0            1.97                                                          16:1 2OH        3.51                                                          17:0 10 methyl  1.18                                                          18:0 ISO        4.17                                                          19:0 ANTEISO    0.2                                                           ______________________________________                                    

REFERENCE LIST

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2. T. R. Shieh et al., Applied Microbiology, Vol. 16 (9), p.1348-1351,1968.

3. Laurent Seaueilha et al., J. Agri. Food. Chem., Vol. 41, p.2451-2454, 1993.

4. Marisa K. Chelius et al., Applied Microbiology and Biotechnology,Vol. 41, p. 79-83, 1994.

5. V. C. Nair et al., Applied Microbiology and Biotechnology, Vol. 34,p. 183-188, 1990.

6. Seong Jun Yoon et al., Enzyme and Micorbial Technology, Vol. 18, p.449-454, 1996.

7. Wim van Hartingsveldt et al., Gene, Vol. 127, p. 87-94, 1993.

8. Christel Lambrechts et al., Biotechnology Letters, Vol. 14 (1), p.61-66, 1992.

9. David B. Mitchell et al., Microbiology, Vol. 143, p. 245-252, 1997.

10. U.S. Pat. No. 5,217,959.

We claim:
 1. An isolated microbe T16-4, which is classified asStreptomyces thermonitrificans and deposited in the China Center forType Culture Collection (CCTCC), Wuhan University, Luo Jia Shan, Wuhan,the People's Republic of China on Sep. 29, 1997 with the accessionnumber of CCTCC M97007.
 2. An isolated microbe T17-1, which isclassified as Streptomyces thermonitrificans and deposited in the ChinaCenter for Type Culture Collection (CCTCC), Wuhan University, Luo JiaShan, Wuhan, the People's Republic of China on Sep. 29, 1997 with theaccession number of CCTCC M97008.
 3. An isolated microbe T17-2, which isclassified as Streptomyces thermovulgaris and deposited in the ChinaCenter for Type Culture Collection (CCTCC), Wuhan University, Luo haShari, Wuhan, the People's Republic of China on Sep. 29, 1997 with theaccession number of CCTCC M97009.
 4. An isolated microbe T19-3, which isclassified as Streptomyces sp. and deposited in the China Center forType Culture Collection (CCTCC), Wuhan University; Luo Jia Shan, Wuhan,the People's Republic of China on Sep. 29, 1997 with the accessionnumber of CCTCC M970012.
 5. An isolated microbe T24-3, which isclassified as Streptomyces diastaticus and deposited in the China Centerfor Type Culture Collection (CCTCC), Wuhan University, Luo Jia Shan,Wuhan, the People's Republic of China on Sep. 29, 1997 with theaccession number of CCTCC M970013.
 6. An isolated microbe T26-6, whichis classified as Streptomyces sp. and deposited in the China Center forType Culture Collection (CCTCC), Wuhan University, Luo Jia Shan, Wuhan,the People's Republic of China on Sep. 29, 1997 with the accessionnumber of CCTCC M970014.
 7. An isolated microbe T28-1, which isclassified as Streptomyces sp. and deposited in the China Center forType Culture Collection (CCTCC), Wuhan University, Luo Jia Shan, Wuhan,the People's Republic of China on Sep. 29, 1997 with the accessionnumber of CCTCC M970015.
 8. An isolated microbe T45-1, which isclassified as Streptomyces aurantiogriseus and deposited in the ChinaCenter for Type Culture Collection (CCTCC), Wuhan University, Luo JiaShan, Wuhan, the People's Republic of China on Sep. 29, 1997 with theaccession number of CCTCC M970017.